Chemotaxis assay procedure

ABSTRACT

A chemotaxis assay procedure which is non-destructive of the cells being studied, which permits the ready performance of kinetic or time-dependent study of cell migration from the same sample, and which produces objective measurements includes the steps of:(a)labeling cells with a dye; (b) placing the labeled cells in a first chamber; (c) placing a chemical agent in a second chamber adjacent to said first chamber; (d) separating said first chamber from said second chamber with a radiation opaque membrane, said radiation opaque membrane having a plurality of substantially perpendicular transverse pores therein; (e) stimulating the labeled cells on the side of the membrane closest to said second chamber with electromagnetic radiation of a first wavelength whereby said labeled cells will emit electromagnetic radiation of a second wavelength; and (f) measuring the emitted electromagnetic radiation from the side of the radiation opaque membrane closest to the second chamber; wherein said radiation opaque membrane comprises a film which is not substantially transmissive to at least one of said first and second wavelengths of electromagnetic radiation. The radiation opaque membrane may comprise a dyed film or a film which has at least one radiation blocking layer applied thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/472,490, filedDec. 23, 1999, which is a reissue application Ser. No. 09/159,427 filedSep. 23, 1998, now abandoned, which is a reissue of application Ser. No.08/383,058 filed Feb. 3, 1995 U.S. Pat. No. 5,601,997.

FIELD OF THE INVENTION

This invention relates to a chemotaxis assay procedure and, moreparticularly, relates to an in vitro chemotaxis assay procedure which isnon-destructive of the cell sample and permits kinetic study of thechemotactic response. This invention also relates to a novel radiationopaque membrane for use in the chemotaxis procedure.

BACKGROUND OF THE INVENTION

Chemotaxis is broadly defined as the orientation or movement of anorganism or cell in relation to a chemical agent. Chemotaxis assays,particularly in vitro chemotaxis assays, are widely used procedures inmedical, biological, pharmaceutical and toxicological research. Suchassays are perhaps most widely used to determine the effect of achemical agent on the inflammatory process, either as a stimulant orinhibitor of that process.

The currently used chemotaxis assay procedure derives from thatoriginally developed by S. Boyden in 1962. (See, S. Boyden, TheChemotactic Effect of Mixtures of Antibody and Antigen onPolymorphonuclear Leucocytes, J. Exp. Med. 115: pp. 453-466, 1962).Essentially, the procedure involves placing a suspension of neutrophilsand a chemical agent in two separate chambers, which chambers areseparated by a polycarbonate filter. The neutrophils are typicallyeither human polymorphonuclear neutrophils (“PMN's”) prepared from theperipheral blood of volunteers or PMN's prepared from the peritonealfluid of animals, such as guinea pigs or rabbits.

After a predetermined period of time, the filter is removed and cells onthe filter surface closest to the chamber containing the cell suspensionare carefully removed. The remaining cells on the filter are then fixedand stained. Using a high power microscope, the filter is examined andthe number of cells appearing on the underside of the filter (i.e., theside of the filter closest to the chamber containing the chemical agent)are counted manually. A positive chemotactic response is indicated bythe cells having migrated or “crawled” through the filter to the sideclosest to the chamber containing the chemical agent. Because of thetime required to do so, typically the entire filter is not examined.Rather, representative sample areas are examined and counted.

There are several disadvantages to this procedure. The examination andcounting of the cells on the filter is time-consuming, tedious andexpensive. It is also highly subjective because it necessarily involvesthe exercise of judgment in determining whether to count a cell that hasonly partially migrated across the filter. In addition, the time andexpense associated with examining the entire filter necessitates thatonly representative areas, selected at random, be counted, thusrendering the results less accurate than would otherwise be the case ifthe entire filter were examined and counted.

Perhaps the most important disadvantage in this procedure is that thefixing step kills the cells. That is, the procedure is destructive ofthe cell sample. Thus, in order to determine a time-dependentrelationship of the chemotactic response; that is, a kinetic study, of aparticular chemical agent, it is necessary to run multiple samples foreach of multiple time periods. When one considers that multiple samples,as well as positive and negative controls, are necessary to obtainreliable data, a single chemotaxis assay can produce dozens of filters,each of which needs to be individually examined and counted. The timeand expense associated with a time-dependent study is usuallyprohibitive of conducting such a study using the Boyden procedure.

Alternatives to the Boyden assay have been proposed to overcome some ofthe above disadvantages. See generally, P. Wilkinson, Micropore FilterMethods for Leukocyte Chemotaxis, Methods in Enzymology, Vol. 162,(Academic Press, Inc. 1988), pp. 38-50. See also, Goodwin, U.S. Pat. No.5,302,515; Guiruis et al., U.S. Pat. No. 4,912,057; Goodwin, U.S. Pat.No. 5,284,753; and Goodwin, U.S. Pat. No. 5,210,021. Although thechemotaxis devices and procedures described in these references havesome advantages over the original Boyden procedure and apparatus, theyare not without their shortcomings. For example, all of theseprocedures, like Boyden, require that the filter be removed and thenon-migrated cells wiped or brushed from the filter before the migratedcells can be counted. In addition, most of these procedures requirefixing and staining the cells and none of them permit the kinetic ortime-dependent study of the chemotactic response of the same cellsample.

SUMMARY OF THE INVENTION

I have developed a chemotaxis assay procedure which avoids the abovedisadvantages, which is non-destructive, and which readily permitskinetic study of the chemotactic response. The chemotaxis procedure ofthis invention is simple, quick and inexpensive to perform, producesobjective data, and is usable with a variety of different cell types.

Basically, the non-destructive chemotaxis assay procedure comprises thesteps of:

-   -   a) labeling cells with a dye;    -   b) placing the labeled cells in a first chamber;    -   c) placing a chemical agent in a second chamber adjacent to said        first chamber;    -   d) separating said first chamber from said second chamber with a        radiation opaque membrane, said radiation opaque membrane having        a plurality of substantially perpendicular transverse pores        therein;    -   e) stimulating the labeled cells on the side of the membrane        closest to said second chamber with electromagnetic radiation of        a first wavelength whereby said labeled cells will emit        electromagnetic radiation of a second wavelength; and    -   f) measuring the emitted electromagnetic radiation from the side        of the radiation opaque membrane closest to the second chamber;        wherein said radiation opaque membrane comprises a film which is        not substantially transmissive to at least one of said first and        second wavelengths of electromagnetic radiation.

In another aspect, the invention comprises a radiation opaque membranefor used in a chemotaxis assay procedure wherein cells labeled with adye are stimulated with electromagnetic radiation of a first wavelengthwhereby the cells will emit electromagnetic radiation of a secondwavelength, said radiation opaque membrane comprising a film which isnot substantially transmissive to at least one of said first or secondwavelengths of electromagnetic radiation, said radiation opaque membranehaving a plurality of substantially perpendicular transverse porestherein.

These and other aspects of the invention will become apparent upon areading of the following detailed description of the embodiments, withreference to the drawings, and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a preferred apparatus used in carryingout the present procedure.

FIG. 2 is an enlarged, sectional view of the apparatus of FIG. 1 as seenalong line 2—2 of FIG. 1.

FIG. 3A is a simplified schematic view, in cross-section, of cellsmigrating across one embodiment of the radiation opaque membrane of thepresent invention.

FIG. 3B is a simplified schematic view, in cross-section, of cellsmigrating across another embodiment of the radiation opaque membrane ofthe present invention.

FIGS. 4-7 are graphs of fluorescence units vs. incubation time of thechemotaxis data generated by the Examples.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Although not critical to the present invention, a description of thepreferred apparatus for use in carrying out the chemotaxis procedure ofthis invention is included because it is believed to be helpful inillustrating the advantages of this invention over the prior art. It isto be expressly understood, however, that any number of devices may beused in carrying out the present procedure and the invention is notlimited to the use of any particular apparatus, except as set forth inthe appended claims.

With reference first being made to FIGS. 1 and 2, the preferredapparatus comprises a multi-well culture plate which is widely availablefrom a variety of commercial sources. This type of apparatus is commonlyemployed to study the effects of chemical agents on cell growth. As seenin FIGS. 1 and 2, the apparatus comprises a plate 20 having a pluralityof spaced-apart wells 22. Each well 22 is provided with an insert 24adapted to fit inside the well. In the parlance of this specification,the interior of the insert comprises one chamber and the exterior of theinsert comprises a second chamber. The size, shape and number of wells22, inserts 24, and plate 20 are not critical to this invention.

For purposes of this invention, the bottom of the insert 24 has beenprovided with a radiation opaque membrane 10 of this invention, whichseparates the two chambers. The radiation opaque membrane 10 may beattached to the bottom of the insert by any conventional means, such asglue or other adhesive, heat welding, ultrasonic welding, etc. Inpractice, the labeled cells are placed in the insert 24 and the chemicalagent is placed in the well 22. The chemotactic reaction will cause thelabeled cells to migrate or “crawl” from the chamber 24 to chamber 22,through the pores 16 in the radiation opaque membrane 10, asparticularly shown in FIGS. 3A and 3B.

As seen in FIG. 2, a space 28 is created between the radiation opaquemembrane 10 and the bottom of the well 22. A distance of about 1 mmbetween the bottom of well 22 and the radiation opaque membrane 10 isgenerally sufficient to permit the free migration of cells across theradiation opaque membrane. The space 28 may be conveniently created byproviding the insert 24 with stand-offs 26, which may take anyconvenient form or shape (e.g. legs, bosses, flange, etc.). When usingstand-offs, care should be taken not to isolate the fluid in space 28from the remainder of the fluid in the well 22, which would tend tocreate a separate concentration gradient in the space 28. Alternatively,the space 28 may be created by suspending the insert 24 within the well22 by the use of, for example, radial projections 27 which rest on thesurface of plate 20 as shown in FIGS. 1 and 2.

At predetermined periods, the quantum of cells that have migrated acrossthe radiation opaque membrane will be determined by first exciting orstimulating the labeled cells on the side of the radiation opaquemembrane 10 closest to the chamber 22 and measuring the radiationemitted by those labeled cells. With the preferred apparatus illustratedin FIGS. 1 and 2, this step would comprise stimulating and measuring theradiation from below the radiation opaque membrane 10, that is, throughspace 28. It will be understood by those skilled in the art that it ispreferred that at least the chamber through which the stimulation andmeasurement of radiation will take place is substantially transparent toboth the radiation being measured and any radiation needed to excite orstimulate the dye used to label the cells. In the preferred embodiment,the apparatus is made of a clear, transparent material, such aspolystyrene, polycarbonate, LUCITE®, glass, etc.

The device 30 used to stimulate the cells and measure the emittedradiation will, of course, depend on the dye used to label the cells andthe type of apparatus used for the assay procedure. For example, if theplate apparatus of FIGS. 1 and 2 is used, a fluorescent plate reader,such as a Cytofiuor™ 2300 (Millipore Corp., Marlborough, MA), can beused to advantage. The radiation opaque membrane 10 will substantiallyprevent either the stimulation of the cells in chamber 24 or thetransmission of radiation from the cell sample in chamber 24 into thespace 28, or will prevent both. Accordingly, the radiation measured willprovide a direct quantitative measure of the number of cells that havemigrated across the radiation opaque membrane 10 from chamber 24 tochamber 22.

It will be appreciated by those skilled in the art that neither insert24, nor radiation opaque membrane 10, nor the non-migrated cells adheredto it, need be removed prior to measuring the radiation corresponding tothe migrated cells. This permits repeated measurements of thechemotactic response of the same cell sample, thus permitting simple andrapid quantitative determinations in a kinetic, or time-dependent,profile of the chemotactic response with a minimum number of testsamples. In addition, the devices used to measure the radiation, such asplate readers or spectrophotometers, are highly sensitive and accuratepieces of equipment and provide objective data corresponding to thenumber of migrated cells. This is a distinct advantage over the priorart procedures which rely upon subjective physical examination under amicroscope.

As mentioned above, the chemotaxis assay of this invention can be usedwith a variety of cell types. Examples include, but are not limited to,macrophages, eosinophils, fibroblasts, endothelial cells, epithelialcells, PMN's, tumor cells and prokaryotic organisms. The only practicallimitations on the cell type are its ability to exhibit a chemotacticresponse and its ability to be labeled.

In accordance with the present invention, the cell sample is labeledwith a fluorescent dye. The process of labeling cells with dyes is wellknown, as is the variety of fluorescent dyes that may be used forlabeling particular cell types. See e.g. R. Haugland, Handbook ofFluorescent Probes and Research Chemicals, Molecular Probes, Inc.(1989). A particularly preferred fluorescent dye for use with an HL-60cell line (ATCC No. CCL 240) in the present invention is Di-I (MolecularProbes, Inc.; Eugene, OR).

It should be mentioned here that, in theory, non-fluorescent dyes may beused in the present invention. At the present time however, there are noknown devices that can be used to measure the transmitted light frommigrated cells to the exclusion of the transmitted light from thenon-migrated cells. Accordingly, the practical utility of usingnon-fluorescent dyes in the present invention awaits the discovery orinvention of such a device.

A particularly novel aspect of the present invention is the use of aradiation opaque membrane which is not substantially transmissive to atleast the wavelength of electromagnetic radiation used to stimulate thelabeled cells or the wavelength of electromagnetic radiation emitted bythe labeled cells. Preferably, the radiation opaque membrane is notsubstantially transmissive to both wavelengths of electromagneticradiation, which would protect against excitation of non-migrated cellsand would also prevent transmission of radiation emitted by anynon-migrated cells that may, nevertheless, become stimulated. It may beadvantageous in certain situations, such as for example where mixed celltypes and multiple labeling dyes are used, to selectively block eitherthe excitation wavelength or the emission Wavelength. Because theradiation opaque membrane is porous, it will be impossible to completelyblock all transmission of radiation across the radiation opaquemembrane, simply because some radiation will be transmitted through thepores in the radiation opaque membrane. In practice, however, thequantum of radiation so transmitted will be relatively constant andnegligible in terms of the quantum of radiation radiating from themigrated cells. Generally speaking, however, the radiation opaquemembrane (absent any pores) should have a blocking efficiency of atleast approximately 95%. That is, the membrane should be capable ofblocking at least approximately 95% of the intended radiation, eitherthe radiation used to stimulate the cells, the radiation emitted by thelabeled cells, or the combined stimulation and emission radiation.

In accordance with the present invention, such membranes permit themeasurement of radiation emitted from the labeled cells that havemigrated through the radiation opaque membrane without interference fromradiation emitted from the labeled cells that have not migrated, withoutthe need to remove the non-migrated cells from the radiation opaquemembrane. This is a significant advantage of the present invention overthe prior art procedures, not only because it avoids the tedious stepsof removing the filter and scraping the non-migrated cells from thefilter, but also because it is non-destructive of the cell sample andthus permits repeated measurements of the same test sample at differenttime intervals.

The radiation opaque membrane itself may be of any convenientconstruction, so long as it has the properties mentioned above. Ingeneral, the radiation opaque membrane 10 comprises a non-fibrous film12 of polyester, polycarbonate, polyethylene terephthalate, polylacticacid, nylon, etc. Depending on the type of film used, the film may bedyed to obtain the radiation blocking properties discussed above. Inlieu of or in addition to using a dyed film, one or more radiationblocking layers 14 may be applied to the film by any conventionalprocess suitable for the particular film and blocking layer(s) beingused, such as coating under vacuum, layer transfer, sputtering, spincoating, vacuum deposition, etc. The thickness of the radiation opaquemembrane 10 is not critical to the invention. Membranes having athickness in the range customarily used in the art are suitable for useherein.

As already noted, the radiation opaque membrane must have a plurality ofpores 16 disposed substantially perpendicular to the plane of theradiation opaque membrane to permit the migration of cells across theradiation opaque membrane. The diameter of the pores is not particularlycritical and, to a large extent, depends upon the size of the cellsbeing studied. Generally speaking, the pores 16 must be of such diameterto prevent the cells from passively traversing the radiation opaquemembrane while at the same permitting the cells to actively “crawl”through the radiation opaque membrane. It is readily within the skill ofthe ordinary artisan to determine the appropriate pore size for aparticular chemotaxis assay without undue experimentation. Pores ofsuitable size can be provided in the film by any known process, such asatomic etching. If a radiation blocking layer(s) is to be applied to thefilm, it may be done either before or after the pores have beenprovided.

EXAMPLES Cell Sample

The cell line HL-60 (ATCC No. CCL 240) was maintained in logarithmicgrowth phase as a suspension culture at about 10⁶ cells/mL. in RPMI 1640medium (Mediatech Cellgrow, Fisher Scientific, Pittsburgh, PA.)supplemented with 20% (volume by volume) fetal bovine serum. (HycloneLaboratories, Salt Lake City, UT). The cells were differentiated intomature myelocytes and neutrophils by incubating the cells for 48 hoursat 37° C. in media containing 1.5% (volume by volume) dimethylsulfoxide.

Cell Labeling

Following the treatment with dimethylsulfoxide, the cells were incubatedwith 50 μM Di-I fluorescent dye (Molecular Probes, Inc., Eugene, OR) atroom temperature for 0.5-2 hours. The cells were then washed with Hanks'Balanced Salt Solution (“HBSS”) (Sigma Chemical Co., St. Louis, MO.) andre-suspended in HBSS to achieve a cell concentration of 10⁶ cells/mL.The fluorescence of 0.5 mL. of cell suspension was measured in aCytofluor™ 2300 fluorescent plate reader (Millipore Corp., Marlborough,MA.).

Membrane Preparation

Membrane 1: Polycarbonate film having a plurality of pores of 8 μmdiameter were coated with four molecular layers of carbon and one layerof an admixture of gold and palladium in a vacuum evaporator. Theresulting radiation opaque membrane had a thickness of about 10 μm andwas approximately 97% efficient in blocking the combined stimulation andemission radiation. 6 mm disks of the radiation opaque membrane wereglued to the bottom of inserts similar to the Millicell HA-12 mm(Millipore Corp.) or the Transwell-6.5 mm (Costar Corp., Cambridge, MA.)inserts with clear silicone rubber cement.

Membrane 2: A non-porous polyester film (18 μm thick) containing a bluedye (Aquired Technology Inc., Alpharetta, GA.) was subjected to atomicetching to produce a 10 μm thick radiation opaque membrane containing aplurality of pores of 8 μm diameter having a combined radiation blockingefficiency of approximately 99%. 6 mm disks of the radiation opaquemembrane were glued to the bottoms of inserts as with membrane 1.

Test Procedure

Each insert equipped with the either membrane 1 or membrane 2 wereplaced in a well of a 24-well culture plate (Falcon, Fisher Scientific).0.5 mL of labeled cell suspension was placed inside each insert. Theplate was incubated for 30 minutes at 37° C. to allow the cells tosettle on the radiation opaque membrane. The fluorescence of each wellwas then measured with the Cytofluor™ 2300 to obtain a zero timereading. 0.5 mL of either N-formyl methionyl leucyl phenylalanine(“f-MLP”) (Sigma Chemical Co.) or HBBS was then added to each well. Thefluorescence in each well was then measured at periodic time intervalsusing the Cytofluor™ 2300 at sensitivity setting 4. Results usingmembrane 1 are reported in Tables 1 and 2 and graphically illustrated inFIGS. 4 and 5. Results using membrane 2 are reported in Table 3 andgraphically illustrated in FIGS. 6 and 7.

TABLE 1 Well Num- Test Solutions Fluorescence ber well/insert 0 hr. 1hr. 2 hr. 3 hr. 4 hr. 5 hr. 1 HBSS/HBSS 546 757 862 922 927 904 2HBSS/f-MLP¹ 383 1046 1355 1433 1370 1359 3 f-MLP²/f-MLP 706 654 708 732728 753 4 f-MLP³/f-MLP 467 412 435 460 447 454 5 Blank 130 124 125 125125 125 6 Blank 132 127 127 128 127 126 7 Blank 131 127 127 128 127 1268 Blank 128 124 126 125 127 125 9 Blank 129 125 126 126 126 125 10 Blank130 127 127 127 128 127 11 Blank 135 133 132 132 132 132 12 Blank 130126 125 126 125 125 13 Blank 132 128 129 130 129 128 14 Blank 134 141136 139 136 137 15 Blank 137 134 133 134 134 132 16 Blank 136 131 132133 132 132 17 Blank 135 134 132 134 131 132 18 Blank 137 132 131 132132 133 19 Blank 136 132 131 132 132 133 20 Blank 139 135 132 135 134135 21 Blank 141 135 136 138 136 137 22 Blank 140 137 137 138 136 137 230.5 mL cells 9999³ 9999 9999 9999 9999 9999 24 0.5 mL cells 9999  99999999 9999 9999 9999 Notes: ¹Conc. = 10⁻⁷ M ²f-MLP added to cellsuspension immediately before start of experiment. ³Fluorescence wasgreater than measurable at selected sensitivity setting.

TABLE 2 Well FLUORESCENCE Num- Test Solutions 0 15 30 60 90 120 150 berinsert/well min. min. min. min. min. min. min. 1 HBSS/f-MLP¹ 2927 41954475 4642 4761 4801 4788 2 HBSS/f-MLP 2895 4165 4400 4539 4642 4681 46553 HBSS/f-MLP 2631 3398 3584 3645 3728 3759 3728 4 HBSS/f-MLP 2594 34463707 3813 3932 3988 3999 5 HBSS/f-MLP 2515 3388 3594 3614 3717 3759 37706 f-MLP²/F-MLP 2854 2675 2721 2721 2783 2783 2783 7 HBSS/HBSS 2558 26832736 2783 2862 2886 2911 8 HBSS/HBSS 2862 2977 3028 3053 3114 3132 31059 HBSS/HBSS 3105 3194 3221 3220 3294 3313 3294 10 HBSS/HBSS 2377 26602767 2846 2927 2952 2960 11 Blank 165 163 162 163 160 160 160 12 Blank166 163 164 162 161 160 657 13 Blank 166 163 163 163 158 161 157 14Blank 166 166 163 163 163 162 159 15 Blank 162 160 160 159 156 157 15616 Blank 163 160 159 159 156 157 153 17 Blank 162 161 160 159 158 156156 18 Blank 164 161 162 159 158 151 147 19 Blank 163 161 162 158 158158 154 20 Blank 162 161 160 159 158 153 145 21 Blank 168 166 165 163163 163 160 22 Blank 165 164 163 159 160 151 151 23 Blank 171 168 168164 150 166 162 24 Blank 172 170 169 153 151 162 162 Notes: ¹Conc. =10⁻⁸ M ²f-MLP added to cell suspension immediately before start ofexperiment

TABLE 3 Well Test Solutions Fluorescence Number insert/well 0 hr. 0.5hr. 1 hr. 2 hr. 3 hr. 1 HBSS/HBSS¹ 1079 1378 1586 1770 1810 2 HBSS/HBSS 891 1058 1194 1351 1421 3 HBSS/HBSS  940 1221 1382 1533 1617 4 Blank 178 169 169 167 166 5 f-MLP²/f-MLP  961 1245 1390 1564 1711 6 0.3 mLcells 9999 9999 9999 9999 9999 7 HBSS/f-MLP³ 1055 1770 2066 2351 2536 8HBSS/f-MLP 1064 1454 1846 2143 2292 9 HBSS/f-MLP 1097 1775 2185 24112432 10 Blank  178 187 196 191 190 11 f-MLP/f-MLP 1049 1277 1413 15381582 12 0.3 mL cells  9999⁴ 9999 9999 9999 9999 13 HBSS/HBSS⁵ 1425 14911577 1682 1735 14 HBSS/HBSS 1359 1454 1491 1551 1645 15 HBSS/HBSS 13401386 1478 1582 1650 16 Blank  179 172 176 178 171 17 f-MLP/f-MLP 11871181 1516 1622 1673 18 0.4 mL cells 9999 9999 9999 9999 9999 19HBSS/f-MLP 1277 1573 1701 1836 1851 20 HBSS/f-MLP⁶ 1228 5928 6063 63426504 21 HBSS/f-MLP 1242 1207 1830 1931 1969 22 Blank  176 171 169 167166 23 f-MLP/f-MLP 1231 1325 1454 1541 1604 24 0.4 mL cells 9999 99999999 9999 9999 Notes: ¹Transwell-type inserts used for wells 1-12.²f-MLP added to cell suspension immediately before start of experiment.³Conc. = 2 × 10⁻⁸ M ⁴Fluorescence greater than measurable at selectedsensitivity setting. ⁵Millicell-type inserts used for wells 13-24.⁶Insert leaked

1. A non-destructive chemotaxis assay procedure comprising the steps of:a) labeling cells with a fluorescent dye; b) placing the labeled cellsin a first chamber; c) placing a chemical agent in a second chamberadjacent to said first chamber, said chemical agent being capable ofinducing migration of said labeled cells from said first chamber to saidsecond chamber; d) separating said first chamber from said secondchamber with a radiation opaque membrane, said radiation opaque membranehaving a plurality of substantially perpendicular transverse porestherein; e) stimulating the labeled cells on the side of the membraneclosest to said second chamber with electromagnetic radiation of a firstwavelength whereby said labeled cells will emit electromagneticradiation of a second wavelength; and f) measuring the emittedelectromagnetic radiation from the side of the radiation opaque membraneclosest to the second chamber; wherein said radiation opaque membranecomprises a film which is not substantially transmissive to at least oneof said first and second wavelengths of electromagnetic radiation. 2.The procedure of claim 1, wherein the fluorescent dye is Di-I.
 3. Theprocedure of claim 3, wherein the radiation opaque membrane comprises apolyester film containing a blue dye.
 4. The procedure of claim 1,wherein the radiation opaque membrane comprises a polycarbonate filmcoated with four layers of carbon and one layer of an admixture of goldand palladium.
 5. The procedure of claim 1, wherein step (f) comprisesmeasuring the electromagnetic radiation with a fluorescent plate reader.6. The procedure of claim 1, further comprising the step of repeatingsteps (e) and (f) at least once at a predetermined time interval.
 7. Theprocedure of claim 6, wherein the dye comprises a fluorescent dye. 8.The procedure of claim 7, wherein the fluorescent dye is Di-I.
 9. Theprocedure of claim 8, wherein the radiation opaque membrane comprises apolyester film containing a blue dye.
 10. The procedure of claim 8,wherein the radiation opaque membrane comprises a polycarbonate filmcoated with four layers of molecular carbon and one layer of anadmixture of gold and palladium.
 11. The procedure of claim 7, whereinthe step (f) comprises measuring the electromagnetic radiation with afluorescent plate reader.
 12. The procedure of claim 6, wherein the filmhas a radiation blocking efficiency of at least approximately 95%. 13.The procedure of claim 1, wherein the film has a radiation blockingefficiency of at least approximately 95%.
 14. The procedure of claim 13,wherein the film has a radiation blocking efficiency of at leastapproximately 97%.
 15. A chemotaxis assay procedure comprising measuringthe migration of cells across a radiation opaque membrane, wherein saidprocedure is non-destructive of said cells.
 16. A procedure of claim 15when said cells are selected from the group consisting of macrophages,eosinophils, fibroblasts, endothelial cells, epithelial cells, PMN's,tumor cells and prokaryotic organisms.
 17. A procedure of claim 15including the step of inducing migration of said sample across saidmembrane.
 18. A procedure of claim 17 including the step of providing achemotactic agent on one side of said membrane.
 19. A procedure of claim18 including the steps of detecting sample presence on said one side ofsaid membrane.